Understanding multidrug resistance in Staphylococcus aureus: The role of efflux pumps and key protein structures

Mohammad Abuzar Shaikh¹, Foorkan Fakki²

Introduction

One of the defining moments in medicine in the 20th century was the development of antibiotics for the treatment of various infections. However, the first germs exhibiting antibiotic resistance were described not long after they were first used in clinical settings. But since, the development of new antibiotics has coincided with an ongoing increase in the number of microbial strains that are resistant to them as well as a diversity of strategies employed by bacteria to evade their fatal effects. Today, each class of widely used antibiotics has at least one documented mechanism of resistance [1]. Additionally, many bacterial species exhibit phenotypes that are multi-resistant. Numerous of these multidrug-resistant (MDR) bacteria can result in infections that are fatal, which makes them a serious issue for both the community and the hospital [2, 3].

One of the main bacterial pathogens, Gram-positive Staphylococcus aureus, can cause infections that are mild to fatal [4]. S. aureus exhibits a surprising range of resistance pathways along with its potential pathogenicity [5]. The MRSA i.e methicillin-resistant S. aureus strains, which have become increasingly isolated from the general population and are resistant to every type of β-lactam antibiotics, are a serious issue. For many years, these strains were a major cause of outbreaks in nosocomial settings [6].

There are many ways that bacteria can resist antibiotics, including degradation or alteration of the antibiotic, switching the antibiotic's bacterial target, safeguarding the target, and lowering the intracellular concentration of the antibiotic through either reduced permeability to cell wall or antibiotic efflux from the cell. Resistance mediated by efflux, in contrast to the other recognised mechanisms, has received less attention [7].

S. aureus is a persistent pathogen that silently persists as our natural flora but occasionally poses a threat to our lives. Its multi-drug resistance phenotype makes it one of the most unbeatable pathogenic bacteria in the history of antibiotic treatment, in addition to its capacity to outsmart human immune system. Almost every antibiotic created since the 1940s was defeated by it. The first MRSA was discovered in clinical isolates of S. aureus in 1961 [98]. Methicillin and vancomycin resistance are the two most notable antibiotic resistances that Staphylococcus aureus has attained. A special staphylococcal mobile genetic element facilitated the interspecies transfer of the mecA gene from an ancestral Staphylococcus species to S. aureus, resulting in methicillin resistance. By horizontally transferring a plasmid-born vanA-gene transposon from vancomycin-resistant Enteriococcus to S. aureus across the genus barrier, vancomycin resistance was attained. The other kind of vancomycin resistance, known as VISA, is brought about by adaptive mutations that are integrated into the genes that control the physiology of bacterial cells [8,9]. Comparative genomics of paired S. aureus isolates has been used in several studies to identify mutations that arise in the resistant strain relative to the parent strain. One of the most notable of these studies was conducted by Mwangi et al., which found that accumulating mutations in the increasingly resistant strain were linked to increasing vancomycin resistance in successive clinical isolates of S. aureus [9].

The topic has, however, become more popular as we have come to understand that a large number of efflux pumps have the ability to remove from the cell a variety of different classes of antimicrobial compounds, which promotes the emergence of resistant strains phenotypes [8]. Drugs may be "accidental substrates" of these transporters due to natural involvement of efflux system in bacteria's ability to eliminate harmful endogenous metabolites, secrete virulence inhibitors, and respond to stress [9, 10].

Efflux pump

When tetracycline resistance in enterobacteria was found to be a mechanism in 1980, antibiotic efflux was first identified. Over the years, bacterial resistance to almost all antibiotics has been associated with efflux mechanisms. The zones of inhibition (MIC) of substrate antibiotics is typically two to eight times higher for bacteria overexpressing an efflux pump than for a susceptible strain of that species [11].

Efflux system in bacteria

Bacterial efflux systems can either be specialized, extruding only one antibiotic or class of antibiotics, or they can be multidrug-resistant efflux pumps, capable of extruding numerous classes of antimicrobial chemicals. The major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, the multidrug and toxic compound extrusion (MATE) family, the resistance-nodulation-cell division (RND) superfamily, and the adenosine triphosphate (ATP)- binding cassette (ABC) superfamily are the five families that these MDR efflux systems are divided into based on their energy requirements and structural characteristics. The transporters of the first four families are secondary transporters that drive the extrusion of their substrates by an antiport H+: drug mechanism, with the exception of the family MATE, which may also take energy from the sodium membrane gradient. In contrast, the ABC superfamily of transporters are the main transporters that propel the ejection of their substrates using ATP as given in figure 1 [12].

Antimicrobial resistance mediated by efflux pump

Changes in drug targets, structural alterations or drug degradation, reduced outer membrane proteins permeability to stop medications to enter cells, and improved transporters are the basis for antimicrobial resistance mechanisms.to lower medication concentrations inside cells. It was previously thought that protein of outer membrane and efflux pumps did not work together to reduce intracellular drug concentrations. In Burkholderia thailandensis, a recent study discovered a connection of efflux pumps and the membrane permeability barrier [14]. In reality, efflux pump overexpression is an important factor in the development of MDR as well as antimicrobial resistance. The creation of efflux pump inhibitors depends critically on our knowledge of the chemical structures of pumps and the important drugbinding sites on those molecules. Following a thorough study of the architectures of various efflux pump families, it will be briefly described [15, 16].

Efflux pumps role in virulence and formation

It has been demonstrated that bile salts and other antimicrobials produced from the host facilitate colonisation and enhance bacterial adaptation to the host digestive system through efflux [17]. The RND Efflux pump AcrAB-TolC, which is mostly implicated in drug efflux in E. coli, can also pass on bile salt resistance [18]. Complex microbial communities known as biofilms adhere to many surfaces, including implanted gadgets like urine catheters. It is commonly known that bacteria that have formed biofilms are more susceptible to antibiotics than germs in the water. In various bacterial species, the connection between the antimicrobial resistance of biofilm and EPs has been documented [19].

Multidrug-resistant efflux pumps encoded by chromosomes

NorA

On S. aureus NorA is the extensively researched efflux systems drug efflux pump. A resistant isolate of fluoroquinolone found in a Japan hospital in 1986 was the first to carry the gene NorA that encodes it [20]. With three norA alleles that have been reported to far and nucleotide sequence differences of up to 10%, the norA gene exhibits some genetic variation [21, 22, 23]. The 388 amino acid protein NorA, which is part of the MFS and has 12 transmembrane segments, is identical to the multidrug Bmr efflux pump from B. subtilis in 44% of the ways and to the tetracycline Tet(A) efflux pump from E. coli in 24% of the ways [24, 25].

According to several investigations, NorA can extrude a wide variety of chemically different molecules, including fluoroquinolones (hydrophilic) like ciprofloxacin and norfloxacin, ethidium bromide like dye, and quaternary ammonium (biocide) compounds [26]. Since norA is known to express at a basal level, this lower sensitivity to certain antimicrobial agents can be partially explained [27]. Through the enhanced expression of the norA, efflux mediated by NorA has been linked to greater resistance to fluoroquinolones, biocides, and dyes [26, 29, 30]. Depending on the mutations that have been acquired in the promoter region of gene NorA, this enhanced expression may be either constitutive or inducible [28]. The details of efflux pumps are listed in Table 1.

NorB

The proof for existence of additional mechanisms of efflux in the S. aureus chromosome has been accumulated since the initial experiments in NorA [31, 32]. The NorA and QacA S. aureus efflux pumps, as well as the Blt efflux pumps (41%) and Bmr of B. subtilis (30%), share structural similarities with the efflux pump NorB. It is a 463 amino acid MFS proton-driven efflux pump with 12 segments of transmembrane. NorB imparts resistance to some substrates of NorA, including the hydrophobic fluoroquinolones moxifloxacin and sparfloxacin, the tetraphenylphosphonium biocide and cetrimide, and tetracycline. It also imparts resistance to substrates of non-NorA, including the hydrophilic norfloxacin and ciprofloxacin [33].

NorB may have a role in the pathogenesis of staphylococcal bacteria, according to research using subcutaneous abscess model of mouse that demonstrated its importance for S. aureus competitiveness [34]. According to subsequent investigations from the same team, NorB may have a role in S. aureus reaction to acidic shock and decreased airflow, conditions that led to the overexpression of the norB gene [35, 36].

NorC

The norC chromosomal gene encodes efflux pump norC. Its an MFS protein with 462 amino acids and 12 transmembrane segments that is 61% identical to NorB [37]. NorC is linked to low-level resistance to ciprofloxacin, moxifloxacin, garenoxacin, and the dye rhodamine, as well as hydrophilic and hydrophobic fluoroquinolones [37, 38]. Studies have shown that norC overexpression is necessary to establish low-level resistance because norC wild-type expression does not appear to be adequate to influence the sensitivity to these compounds [37].

Benzalkonium chloride, dequalinium tetraphenylphosphonium, ethidium Cetrimide, benzalkonium chloride, cetyltrimethylammonium, ethidium Benzalkonium chloride, cetyltrimethyl Benzalkonium chloride, cetyltrimethyl

Multidrug-resistant efflux pumps encoded by plasmid

9 QacA/B Diamidines, Biguanidines, bromide, rhodamine 10 Smr ethidium bromide 11 QacG Benzalkonium chloride, bromide 12 QacH ammonium, Proflavin 13 QacJ ammonium, ethidium bromide

MepA

MepA was discovered in research using mutants of S.

aureus with norA disrupted [39]. It was the first multidrug transporter from the MATE family to be described in S.

aureus. It is encoded by the chromosomal mepA gene. 12 transmembrane segments make up this 451amino acid protein, which shares 26% and 21% of its amino acid MFS QacR 46, 47, 48 SMR Not identified 53, 54, 57 SMR Not identified 60 SMR Not identified 61 SMR Not identified 62 NorM of Vibrio parahaemolyticus (MATE) transporter respectively. MepA was discovered to be linked to an Resistant phenotype, which confers Reduced resistance to quaternary ammonium substances like cetrimide, benzalkonium chloride, tetraphenyl-phosphonium, dequalinium, and ethidium bromide dye, as well as to pentamidine, chlorhexidine, and tigecycline, a glycylcycline antibiotic. Norfloxacin and ciprofloxacin were found to be operon contains the mepA gene. The encoded MepR protein shares similarities with MarR family regulatory proteins, according to sequence analysis. MepB and other proteins with recognized functions were not found to have any significant similarities, and there was no correlation between the MepB gene and Resistant Multidrug phenotypes [39].

mdeA

An open expression library of the genome of S. aureus revealed the chromosomal mdeA gene, which encodes the efflux pump MdeA. MdeA is a member of the MFS, has 479 amino acids, and 14 transmembrane segments, and uses the proton motive force to energise the transport of its substrates. MdeA and the efflux pump LmrB from B. subtilis, EmrB from E. coli, and QacA from S. aureus share 37% of their genetic makeup. The overexpression of mdeA has been connected to increased resistance to the biocides (e.g. benzalkonium chloride, dequalinium, and tetraphenylphosphonium), the dye (e.g. ethidium bromide), and the antibiotics (e.g. virginiamycin, novobiocin, mupirocin, and fusidic acid). Mutations that occur in the mdeA promoter area can cause the overexpression of mdeA, but they only slightly increase the phenotype of MDR resistance [41]. The norfloxacin and ciprofloxacin (fluoroquinolones) are less active substrates of this pump, according to a subsequent study [42].

SepA

As an efflux pump, the SepA protein, which is coded by the chromosomal sepA gene, confers Reduced antiseptic resistance (e.g. benzalkonium chloride, chlorhexidine gluconate, and the acriflavine dye. This transporter has four putative transmembrane segments and 157 amino acids, which are shared by SMR family transporters. SepA lacks the conserved motifs of this family even though several residues essential for the transport specificity and the antiport H+: drug are present in a different place., which raises the possibility that it may be a member of an unidentified family of transporters [43].

SdrM

The chromosomal gene sdrM codes for the efflux pump SdrM. The pumps NorB and QacA S. aureus Resistant efflux share 23% and 21% of their identity with SdrM, respectively. Sequence research suggests that SdrM, which has 14 transmembrane segments, maybe a member of the MFS. By encouraging an energy-dependent outflow of these substances, it was demonstrated that this efflux pump is linked to Reduced resistance to ethidium bromide, acriflavine, and norfloxacin [44].

LmrS

lincomycin resistance protein S. aureus i.e LmrS was recently characterized by Floyd and colleagues. t is 39% LmrB and 25% identical to the efflux pumps FarB (Neisseria gonorrhoeae) and EmrB (E. coli) The improved linezolid resistance, sodium dodecyl sulphate, tetraphenyl phosphonium chloride, chloramphenicol, and trimethoprim, was attributed to LmrS, a 480 amino acid MFS protein with 14 putative membrane-spanning domains [45].

Multidrug-resistant efflux pumps encoded by plasmid

QacA/B

Clinical isolates of S. aureus carries pSK1 plasmid, a resistance-conferring gene to a variety of disinfectants and antiseptics was discovered in the early 1980s [46]. The QacA gene member of the MFS, which has 514 amino acids and 14 transmembrane segments, is encoded by this gene, which was later given the name qacA [47]. Large conjugative plasmids from coagulase-negative staphylococci as well as strains of S. aureus include the qacA gene [48]. From 12 different chemical classes more than 30 mono and divalent cations (lipophilic), including dyes (e.g. ethidium bromide and rhodamine), quaternary ammonium compounds (e.g. tetraphenyl phosphonium, benzalkonium chloride, and dequalinium, diamidines (e.g. DPAI and pentaamine), biguanidines (guanylhydrazones, and chlorhexidine) are used. The proton motive force drives the transport of these substrates through an antiport H+: drug mechanism [49-52].

Smr

The smr efflux pump gene, which confers resistance of ethidium bromide and antiseptic, was discovered in numerous plasmids by several groups in the late 1980s. The determinants identified by these authors as belonging to this gene were given the names ebr, qacC/D, or smr [53-55] but sequencing examination revealed that these all determinants identical. Smr gene, which encodes the efflux pump Smr, can be seen in S. aureus as well as staphylococci (coagulasenegative) in the form of tiny non-conjugative plasmids (pSK89) or big conjugative plasmids (pSK41) [56, 57]. Smr, which comprises 107 amino acids and four transmembrane segments, is a member of the SMR family and uses the proton motive force to energise the transport of toxic chemicals, according to hydropathy research. Comparing this efflux pump to QacA/B, a smaller subset of chemicals cationic dyes (ethidium bromide) and quaternary ammonium compounds (benzalkonium chloride) convey reduced resistance. Smr may undertake efflux, as demonstrated by in vitro transport tests using pure Smr gene and a site-directed mutagenesis, albeit it is unclear whether this is done as an oligomer or monomer as is the case for other efflux pumps in the family of SMR [58, 59].

QacG

S. aureus isolates gathered from the food sector included the efflux pump gene qacG. The plasmid pST94 (2.3 kb)

QacG, a member of the SMR of transporters with one hundred and seven amino acids and four segments of transmembrane [60].

QacH

QacH is 69.2% identical to smr efflux pump. The S. saprophyticus food sector strain contained a 2.4 kb isolated plasmid that contained the determinant qacH for the first time. The efflux pump QacH, which has one hundred and seven amino acids and four segments of transmembrane. and is a SMR member, is encoded by the qacH, which shares 76% of its nucleotides with the smr gene and 70% of its nucleotides with the qacG gene [61].

QacJ

S. aureus, S. simulans, and S. intermedius staphylococcal species all obtained from horses, were discovered to share the qacJ efflux pump gene on a plasmid (2.65 kb). The encoded efflux pump QacJ is 72.5% identical to Smr, and 73.4% and 82.6% identical to QacH and QacG efflux pumps respectively. It is a member of the SMR and has 107 amino acids and four segments of transmembrane. All of these pumps have equal specificities for substrate, imparting same levels of resistance to ethidium bromide, benzalkonium chloride, and cetyltrimethylammonium bromide. This is true even though the QacG/H/J amino acid having different sequences than Smr [62].

Clinical values and significance of efflux pumps

Efflux pump inhibitors are prospective antibacterial medicines because of the extensive distribution of efflux pump gene in bacteria S. aureus and the strong link with resistance to antibiotics in the clinic. Inhibiting efflux pumps is anticipated to have the following effects:

i) Reactivating antibiotics that were previously made ii) Lowering the frequency of occurrence of mutants(resistant); and iii) Lowering the sensitivity of the bacteria, as shown in figure number 2.

Over the last ten years lot of research have been undertaken to find efflux pump inhibitors that are effective against S. aureus, with a particular focus on NorA [63,64]. Reserpine and phenothiazines are two antipsychotic/antihypertensive medications that have been shown to inhibit S. aureus efflux pumps, although their use has been restricted due to their cytotoxicity [65].

Bacteria's natural defensive mechanism against dangerous compounds in their ecological environment is the efflux of antibacterial agents. In order to compete with other bacterial pathogens, eukaryotic hosts, and other bacteria, bacterial pathogens have evolved to withstand a variety of antibiotic compounds. Since efflux pumps are the initial step of defence against antibacterial agents, their inherent function may be a defence against such naturally occurring compounds. While the intrinsic activity function of efflux pumps has primarily been studied in species of Gramnegative and Gram-positive (S. aureus) [66,67]. Drug effluxmediated clinically meaningful resistance in bacteria can include biocides, antibiotics, or both. The extrusion of multiple classes of antibiotics by MDR efflux pumps, like the ones discussed in this review, or efflux systems that can extrude a single class of antibiotics, like the Tet determinants that transmit resistance to tetracycline, are examples of efflux-mediated antibiotic resistance. A multidrug resistance phenotype might arise from the overexpression of MDR efflux pumps because of their promiscuous substrate selectivity, which can increase resistance to many antibiotic classes while simultaneously decreasing susceptibility to biocides [112,113].

Assessment of efflux activity

One must ensure that the methodology used is the most suitable to reveal efflux activity in order to completely determine the function that a particular efflux system plays in antibiotic and/or biocide resistance. The majority of research on efflux-mediated resistance in clinical isolates of S. aureus uses the so-called efflux inhibitors, which are substances that have been shown to impede efflux activity, to lower the antibiotic minimum inhibitory concentration (MIC) [101, 102]. This method is time-consuming and depends on how susceptible the efflux system or systems are to that specific inhibitor, which can vary greatly and for which the cellular mechanism of action is typically still unclear [103-105].

Ethidium bromide, an extensive variety of efflux pump substrate, has been investigated more recently as a means of measuring efflux activity in S. aureus cells. The Ethidium Bromide-Agar Cartwheel Method, which assesses the cells' ability to retain/extrude ethidium bromide after an overnight stay [106], the measurement of ethidium bromide MICs to identify S. aureus strains that exhibit increased efflux activity [107, 108] in the the presence or absence of efflux inhibitors [109, 110], or assays that directly assess the efflux activity using real-time fluorometry [111] are examples of how this substance can be used as a marker for the indirect evaluation of efflux activity.

Regulation and application of efflux pumps related to protein/gene expression

Mechanism of efflux pump regulation

Various regulatory systems and proteins also have a role in controlling efflux pump expression, in addition to exposure to disinfectants or antibacterials. Currently, single MDR pump regulatory proteins are mostly categorised into four groups: MarR, MerR, AraC, and TetR [68]. Both domains ligand-binding and DNA-binding are present in these regulatory proteins. tet genes related to efflux, which provide tetracyclines resistance, are controlled in expression by the TetR, a transcriptional repressor that depends on substrate [69]. Overexpression of the new MATE efflux pump FepA leads to fluoroquinolone resistance. The norfloxacin and ciprofloxacin MIC values in L. monocytogenes are raised as a result of a TetR-type repressor FepR, which controls it [70]. MgrA controls the ABC efflux pump AbcA, the non-MDR tetracycline efflux pump Tet38, and the three S. aureus MDR efflux pumps NorA, NorB, and NorC [113].

For instance, it is known that the regulation systems, AcrR, Rob, EnvR, MprA, MarA, PhoP, and RpoE all have a role in the regulation of AcrAB (E. coli). These regulatory mechanisms are triggered by signals from the environment, such as pH, antimicrobial concentrations, organic solvents, metal ions, growth phase, and oxidative stress [71]. Certain regulators can also alter the transcription of the genes that code for certain MDR efflux pumps in S. aureus. For MepA and QacA/B, which are regulated by MepR and QacR, respectively, this is true. Both of these regulators are sensors that bind to the MDR efflux pumps' substrates and trigger their expression, making them substrate-responsive regulators [113]. MepR is a self-repressive protein that attaches to the motif GTTAG, which is found in the promoter areas of mepR and mepA, as well as to sequences that contain pseudo palindromes. The mepB promoter region is not bound by MepR [114]. MepR binds to the mepA promoter more strongly than it does to the mepR promoter, and the ratio of this binding is most likely different [115].

The gene qacR, which is located just upstream of the genes qacA and qacB, which are transcribed differently from these genes, encodes QacR. QacR is a member of the TetR family of transcriptional repressors and has a helix-turn-helix DNA binding motif that is typical of regulatory proteins [116]. By attaching itself to the qacA promoter, QacR has been demonstrated to be a direct suppressor of the qacA gene's expression [117]. QacR attaches itself to a big inverted repeat that crosses over the qacA/B transcription start sites and is situated directly downstream of the qacA/B promoters [118].

Antibiotic residue detection using efflux pump proteins

Only two regulatory proteins, TetR and TtgR, are utilized to screen for antibiotic residues at this time; proteins related to efflux pump are still being studied for this purpose (belong to TetR family). Utilizing proteins involved in gene transcription, a concentration of low antibiotics might be detected efficiently [72]. Hyerim et al. proposed a bioreporter technique focused on TetR and the TetR promoter for detecting doxycycline using a green fluorescent protein (GFP) gene as a reporter gene. GFP could be expressed in significant numbers in response to 5nM doxycycline [73]. Tetracycline antibiotics may be precisely and promptly determined using an in-vitro indirect ELISA developed by Weber et al. utilizing TetR-tetO, with limit of detection 0.1 and 1.9 ng/mL of doxycycline and tetracycline, respectively [74]. Furthermore, Espinosa-Urgel et al. proposed a unique microbial biosensor centered on TtgR that estimates drug concentrations as low as 22 M with high fluorescence. According to research, drugs such as ceftazidime, ciprofloxacin, and tetracyclines which are P. putida's GFP-fused TtgR, a TtgABC efflux pump transcription regulator, react the most [75].

Antibiotic residue detection using gene expression

Resistant efflux pumps in microorganisms cause them to be naturally or unavoidably resistant to antimicrobial drugs. The efflux pumps' constituent or regulatory proteins, which ensure the bacteria's survival, are encoded by the resistancerelated genes [76]. Antimicrobial resistance can be quickly detected using a variety of efflux pump genes, which can then be successfully confirmed using identification by PCR and determination of MIC. The RND pumps are capable of recognizing various substrates, causing the majority of medicines to be extruded and boosting antimicrobial resistance. Examples include E. coli's Acr pump, A.

baumannii's Ade pump, and P. aeruginosa's Mex pump [77, 78].

Additionally, MICs and high levels of resistance are linked to several single-substrate efflux pumps. These singlesubstrate efflux proteins include the tetracycline-mediated efflux pump TetA/TetO in E. coli [79], the fluoroquinolones (hydrophilic) efflux pump OqxAB from S. enterica and E.

coli, and MacAB the macrolide efflux pumps in E. coli and Mef in S. pneumoniae [80, 81].

Including PDB IDs for various efflux pumps in the review, article is highly significant as it provides researchers with precise structural information required for computational drug design and target-specific studies. These PDB IDs serve as a critical resource for understanding the threedimensional structure and molecular dynamics of efflux pumps, enabling the identification of potential binding sites and the development of inhibitors to overcome multidrug resistance. By consolidating this information in a single table, the article facilitates streamlined access to essential data, promoting further research and innovation in targeting efflux pump proteins for therapeutic intervention. Table 2 and 3.

transporter (K398A mutant) in an open conformation in complex with a single transporter in an outward conformation in complex with a single , a transcription regulator of bound to ethidium bromide Crystal structure of the Secreted Extracellular protein Staphylococcus aureus Solution structure of the B3BP Smr domain DIPHTHERIAE at 2.96 A resolution QacR 2GBY Structure of QacR Multidrug Transcriptional Regulator Bound to Bivalent Diamidine Berenil 7 7D5Q Structure of NorC outwardchain Indian camelid antibody 8 7D5P Structure of NorC camelid antibody 9 4XYD Nitric oxide reductase from Roseobacter denitrificans (RdNOR) 10 4LQE Crystal Structure of MepB 11 3ECO Crystal structure of MepR the Staphylococcus aureus multidrug efflux pump MepA 12 5FFZ S. aureus MepR 13 5FFX S. aureus MepR G34K Mutant 14 4L9N Crystal structure of MepR multidrug resistant S. aureus clinical isolate 15 5J44 A (SepA) from Shigella flexneri 16 7Y58 CryoEM structure of QacA (D411N), an antibacterial efflux transporter from 17 2VKC 18 3NPI Crystal structure of a TetR family regulatory protein (DIP1788) from CORYNEBACTERIUM 19 2UXH TtgR in complex with Quercetin

Conclusions and future perspectives

Resistance to traditional antibiotics has grown significantly since the last few decades, posing a serious danger to antibacterial chemotherapy regimens used to treat a variety of persistent, life-threatening infections. There may someday be no viable antibiotics available for therapy due to the alarming rise in multidrug-resistant bacteria. This situation is referred to as "the post antibiotic age." Antibiotic active efflux, particularly through multidrug efflux pumps, is regarded as a significant mechanism of multidrug resistance [122] NorC 86 - -open NorC 86 -chain Indian NorC 87 MepB 88 MepR 89 MepR 90 MepR 91 A103V mutant from MepR 92 4L9N SepA 93 QacA 94 Smr 95 TetR 96 TtgR 97 for the emergence of resistance to antibiotic, and survival of bacteria in the environment in bacteria. Therefore, it makes sense to suppress bacterial efflux pumps in order to counteract multidrug resistance. By restoring the lost potency that active extrusion via pumps caused in existing antibiotics, efflux pumps inhibition is anticipated for improving their efficacy. Blocking efflux pumps would also lessen bacterial pathogenicity, the emergence of resistance to antibiotic, and bacterial survival in the surrounding [99]. Drug efflux pumps, an important factor in the resistance mechanism, has drawn a lot of interest from researchers working to tackle drug resistance. To improve the clinical efficiency of antibiotic, pathogenic S. aureus, MRSA can be treated with efflux pump inhibitor strategies. Due to these potential consequences, research on efflux pumps has been greatly accelerated, and S. aureus and other bacterial pathogens have benefited from the development of efflux pump inhibitors [100]. To create efficient efflux pump inhibitors, it is required to investigate the inhibitory mechanisms and conduct potential inhibitors testing by invivo studies. Additionally, a deeper comprehension of the design, mechanisms, control and relationship of the efflux pumps with clinical antibiotics resistance would make it possible to create better medications that are less vulnerable to bacterial resistance.

Researchers looking to investigate the structural Aspects and molecular of multidrug resistance will find PDB IDs helpful for important proteins and genes linked to the S. aureus efflux pumps. These structural understanding may help overcome current challenges in the war against antibioticresistant bacteria by enhancing the development of novel treatment approaches and targeted inhibitors. Future researcher can integrate the better understand the molecular and structural mechanisms of resistance by utilizing these PDB IDs, opening the door to more potential and practically applicable approaches.

Acknowledgment

We are thankful to management and principal of AIKTC School of Pharmacy, New Panvel, India for providing the facilities and infrastructure to carry out the research work.

Competing interests

The authors declare no competing interests.

Funding statement

None.

Ethics approval and consent to participate

Not applicable

Data availability

All data generated or analyzed during this study are included in this published article.

References

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